Guanine Nucleotide-sensitive Interaction of a Radiolabeled Agonist with a Phospholipase C-linked P2y-purinergic Receptor

摘要: Abstract Analogs of ATP and ADP produce a guanine nucleotide-dependent activation phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent those P2y-purinergic receptor (Boyer, J. L., Downes, C. P., Harden, T.K. (1989) Biol. Chem. 264, 884-890). This study describes the interaction adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) this putative on purified plasma prepared from erythrocytes. In binding assays performed at 30 degrees C, association rate constant [35S] was 1.1 x 10(7) M-1 min-1 dissociation 3.8 10(-2) min-1. [35S]ADP S bound high affinity (Kd = 6-10 nM) to an apparently homogeneous population sites (Bmax 2-4 pmol/mg protein). analogs (2-methylthio ATP, S, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, beta, gamma-methylene adenosine 5'-triphosphate) inhibited ligand by simple law mass action kinetics single site. The rank order potency for inhibition identical observed these same agonists stimulation ghosts. Guanine nucleotides noncompetitive manner following order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate GTP GDP 5'-O-2-(thiodiphosphate). data are idea that may be used radiolabel linked membranes. addition, radiolabeled agonist is modified nucleotides, providing evidence agonist-induced receptor/guanine nucleotide regulatory protein complex involved P2y-receptor action.